We have recently reported that primitive human hematopoietic progenitors, capable of initiating long-term bone marrow cultures, termed LTC-IC, are maintained when cocultured with stroma but separated from the stromal layer by a 0.4 m microporous membrane in a "stroma-non-contact" culture. Preliminary studies indicate that cytokines together with proteoglycans released by stromal cells are responsible for the LTC-IC maintenance capacity of "stroma-non-contact" cultures. In additional studies we demonstrate a 5-fold increase in LTC-IC number (5 fold expansion) when cultured for 2 weeks in "stroma-non-contact" cultures supplemented with IL3 and the presumed growth inhibitory cytokine MIP-1alpha, suggesting LTC-IC proliferation (self-renewal). However, the number of LTC-IC present after 5-8 weeks in "stroma-non-contact" cultures without (40%) or with MIP- 1alpha+IL3 (100%) is lower than or equal to that in the starting population (40-100% maintenance) suggesting that some LTC-IC may not persist or be conserved over time. These studies led us to hypothesize that long-term expansion of LTC-IC in ex vivo cultures will require conditions that induce (A) increased proliferation (LTC-IC undergoing self-renewing cell divisions) and (B) increased conservation of LTC-IC (persistence of LTC-IC over time). We propose a series of studies to identify additional known growth factors and to characterize further diffusible, large molecular weight stroma-derived factors which induce proliferation and conservation of human hematopoietic stem cells. In SA1, we will develop a single cell deposition assay which can evaluate the fate of individual LTC-IC, i.e. is a single LTC-IC conserved? and did a single LTC-IC proliferate? This assay system will then be used to examine the effect of altered culture condiitons as proposed in SA2 and SA3 on the proliferation and conservation of LTC-IC. In addition, we will develop a more rapid screening assay for LTC-IC proliferation using PKH-26 fluorescence labeling of progenitor populations. In SA2, we propose to examine the effect of presumed growth inhibitory cytokines combined with known growth promoting cytokines on the proliferation and conservation of LTC-IC. Cytokines, such as PF4, IL7 and b-FGF, which interact with primitive progenitors, are present at very low concentrations in stromal cultures and can bind to proteoglycans, will be included. Since preliminary studies indicate that proteoglycans produced by stromal feeders are imperative for LTC-IC maintenance, experiments in SA3 will characterize diffusible proteoglycans present in stroma conditioned media required for LTC-IC proliferation/conservation. Once the nature of the glycosaminoglycan side chains and core protein have been identified, synthetic proteoglycans will be constructed and tested for their LTC-IC proliferation/conservation capacity. Finally, in SA4, we propose to demonstrate that multilineage, long-term repopulating, hematopoietic stem cells have expanded in the presence of the cytokines identified in SA2 and proteoglycans identified in SA2 and proteoglycans identified in SA3 by transplanting retrovirally marked progenitors recovered from ex vivo cultures into preimmune lambs. These efforts provide the foundation for additional studies proposed in Project 2 exploring the lymphoid differentiation capacity of hematopoietic stem cells and for clinical trials employing expanded progenitor populations in transplant therapy for malignant diseases as described in Projects 3-5.